Protein for blocking platelet adhesion

ABSTRACT

A naturally occurring protein isolated from the saliva of the medicinal leech  Hirudo medicinalis  is described which strongly binds to collagen thus acting as an inhibitor of natural platelet adhesion to collagen. The protein has a molecular weight of about 12,000, an acidic isoelectric point and contains six cysteins. The protein was sequenced and the gene was cloned from a  H. medicinalis  cDNA-library. Procedures for producing such polypeptide by recombinant techniques are disclosed. The recombinant and the natural occurring proteins are potent inhibitors of collagen-dependent platelet adhesion and therefore useful for the therapeutic treatment of various conditions related to hard disease and diseases of the circulation system. Furthermore the protein is useful for coating natural or artificial collagen surfaces in order to render them nonadhesive for cells and prevent the activation of cells.

This application is a continuation of U.S. patent application Ser. No.09/936,737 filed Sep. 17, 2001 now U.S. Pat. No. 6,774,107, which claimsthe benefit and national stage of PCT/EP00/02117 filed Mar. 10, 2000.

SUMMARY OF THE INVENTION

A naturally occuring protein isolated from the saliva of the medicinalleech Hirudo medicinalis is described which strongly binds to collagenthus acting as an inhibitor of natural platelet adhesion to collagen.The protein has a molecular weight of about 12,000, an acidicisoelectric point and contains six cysteins. The protein was sequencedand the gene was cloned from a H. medicinalis cDNA-library. Proceduresfor producing such polypeptide by recombinant techniques are disclosed.The recombinant and the natural occuring proteins are potent inhibitorsof collagen-dependent platelet adhesion and therefore useful for thetherapeutic treatment of various conditions related to hard disease anddiseases of the circulation system. Furthermore the protein is usefulfor coating natural or artificial collagen surfaces in order to renderthem nonadhesive for cells and prevent the activation of cells.

FIELD OF INVENTION

In haemostasis or thrombosis, platelets adhere to the cell-extracellularmatrix of an injured vessel and cover the surface of the damaged area.Preventing this important initial step in the pathogenesis of thrombosisand arterial occlusion should be of therapeutic benefit in the effort ofto prevent thrombotic diseases. Collagen is considered to be the mostthrombogenic surface component and has been shown to be a strongstimulant for platelet adhesion, aggregation and the release of theirgranules leading to the recruitment of (Ruggeri, Z. M. et al.; Seminarsin Hematology, 1994, 31, 229-39) additional platelets to this area toform aggregates or a thrombus. The initial contact of the platelets tothe vessel surface is mediated by collagen bound von Willebrand Factor(vWF) and a specific vWF receptor on platelets, the glycoprotein Ib-V-IXcomplex. In addition ADP, epinephrine and circulating clotting factorsdrive the further activation process of platelets while simultaneouslyan increase in thrombin activity contributes to the formation of thecross-linked fibrin clot. Platelet-platelet aggregation supports thisprocess and is mainly driven by fibrinogen as a mediator that bridgescells through the glycoprotein IIb/IIIa receptor.

This normal physiological response is quite critical in the course ofthe pathological process where platelets adhere to collagenes exposendin sclerotic lesions (Van der Rest M. et al.; FASEB Journal, 1991, 5,2814-23) and start to bild-up occlusions. Depending on the location andextent of the occlution severe complications such as myocardialinfarction, stroke, inflammation or pulmonary to embolism may be thesevere outcome of this process.

As a direct acting antithrombotic agent heparin which blocks thethrombin activity, thus preventing the formation of fibrin rich thrombi,is the currently most well known drug used in anti-thromboticinterventions. Heparin is widely used in indications such as: unstableangina and acute myocardial infarction. However despite the wide useseveral severe short comings such as intravenous application,requirement for anti-thrombin-III as a cofactor, reduced affinity forclot-bound thrombin, it's inactivation by several plasma proteins, theoccasional induction of thrombocytopenia and it's biologicalheterogeneity remain unresolved. As a consequence the results of usingheparin in the clinical setting have not been overwelming sofar.

Recent development of low molecular weight heparin has contributed aversion for subcutaneous application, however the therapeutic benefitover the standard heparin has been modest. Unfortunately the sameapplies to the other directly acting antithrombins such as Hirudin,Hirulog and Warfarin. It turned out, that one of the major problemsseems to be related to the increased production of thrombin underantithrombotic treatment (Rao, A. K et al., Circulation, 1996, 94,389-2395).

Other recent strategies have therefore been focussed to the process ofprothrombin activation which is driven by Factor Xa. The major challengeis the design of appropriate inhibitors directed to this factor. Insummary on would therefore argue that the full therapeutic potential ofthis type of intervention has not yet been realized.

Another pannel of therapeutics is represented by the thrombolyticregimens and has been focussed on the development of staphylokinase,streptokinase, urokinase type Plasminogen Activator, tissue typePlasminogen Activator and anisoylated-plasminogen-streptokinaseactivator complex. The differences in time necessary to inducingreperfusion is remarkable different for each of these thrombolyticagents, however the contribution in terms of reducing the overallmortality is equal for all the products. In addition reocclusion orprolonged bleeding are frequent complications. This might be due torelatively low specificity for fibrin and the short plasma half-life ofthese compounds. Currently various application regimens and combinationsof different fibrinolytic principles are tested in order to overcomesome of the current short comings in thrombolytic therapie. Theimprovement which are expected are however rather small.

Recently a new group of patients occured with probelms such as acutethrombotic occlusion and late restenosis due to procedures such asangioplasty, atherectomy, arterial grafting or vessel wall stenting. Thepossible therapeutic interventions comprise anti-platelet,antithrombotic and thrombolytic strategies. Various other agents such asticlopdine acting as ADP antagonists or Calcium ionophore A-23187 andespecially Asprin have a direct influence on the platelet function andhave been suggested or used to prevent or minimize platelet aggregation.The new anti-platelet adhesion substance according to this inventioncould as well help to overcome these clinical complications when appliedduring surgery.

Another complication related to this topic arises if artificial surfacescome in contact with blood, then their is increased tendency to inducethrombotic events by activation of platelets and/or induction ofcoagulation. These effects may cause failure of vascular grafts, cardiacvalves, stents, catheters or any other blood contacting device ormaterial. The ability of the protein disclosed here to createnon-thrombogenic surfaces may therefore be further exploited byimmobilization of this protein to the materials and devices describedabove. Such a treatment should render such materials or devicesbiocompatible and thromboresistant.

Due to the limitations associated with the available antithromboticagents there is an actual need for new alternative strategies andtherapeutics.

BACKGROUND OF THE INVENTION

A potential for future improvements in the treatement of caridvaculardisorders may be contributed by approaches as disclosed in thisinvention which directely interfere with the collagen and/or vWF factorinduced platelet adhesion.

Several novel inhibitors which prevent platelet adhesion are monoclonalantibodies directed to vWF. It has as well been suggested thatglycoprotein IIb/IIIa inhibitors may be beneficial in inhibitingplatelet adhesion

Some of these inhibitors like the monoclonal Ab c7E3 have already beentested clinically while others like the KGD- and RGDF-inhibitors arestill under study. However, the specificity of most of these newinhibitors is not very well studied, thus the spectrum of side effectsthat will be induced by using this inhibitors is still open and deservescarefull examination.

A rich source for the screening of new compounds that interfere withcollagen induced platelet adhesion is given in nature throughblood-sucking animals. Several inhibitors have been isolated from natureas described in the literature: A 65 kD protein called Calin isolatedfrom Hirudo medicinalis (U.S. Pat. No. 5,587,360, WO 92/07005) (Munro,R., et al., Blood Coagulation and Fibrinolysis, 1991, 2, 179-184) and a16 kD (LAPP) protein isolated from the salivary glands of the leechHaementeria officinalis (U.S. Pat. No. 5,324,715). Both proteins havebeen described as aggregation inhibitors as tested in static assays ofcollagen dependent platelet aggregation.

Despite a proven in vitro activity LAPP failed to act in severalwell-established in vivo models (Schaffer L. W. et al.; Arterioscler.Thromb., 1993, 13, 1593-1601) and Connolly T. M. et al.; Thromb.Haemostas., 1993, 69, 589). The soft tick, Omithodoros moubata, alsocontains an antiplatelet protein (Moubatin) which is active inpreventing collagen-stimulated platelet aggregation (Waxman, L. et al.;J. Biol. Chem., 1993, 268, 5445-49). Another inhibitor of plateletaggregation from a blood-sucking bug was disclosed in WO 9309137 byNoeske-Jungblut C. et al. Smith et al. have isolated a 50 kDa proteinfrom snake venom and a 19 kDa protein was isolated from a the saliva ofTriatoma pallidipennis, a blood-sucking bug. The protein was found tocontain a factor that specifically inhibits collagen-induced plateletaggregation. The 19 kDa protein named pallidipin inhibitscollagen-mediated aggregation of platelets in plasma. No inhibition ofaggregation stimulated by other effectors (ADP, thrombin, thromboxane A2mimetic U46619, phorbol ester) was detected. Pallidipin had no effect onplatelet adhesion to collagen but inhibited ATP release from platelets.It interacted reversibly with platelets and may share with collagen acommon target on them. The precise mechanism of action and therapeuticbenefit of this protein is under investigation. Gan et al. describedEchistatin as an inhibitor binding to the fibrinogen receptor GPIIa/IIIb (J. Biol. Chem, 1988, 263, 19827-32).

Despite these exciting developments, the need continues to exist forsupplying further anticoagulants and antithrombin which have increasedefficacy in the inhibition of clot formation, vWF-induced plateletactivation or endothelial cell activation and which may be usedpharmaceutically and produced in commercially feasible quantities.

Since none of the known proteins described sofar has developed into acompound with ideal therapeutic profile the inventors of the presentinvention decided to go ahead with a new screening strategy in order todetect more relevant proteins.

DESCRIPTION OF THE INVENTION

In this invention an inhibitor isolated from H. medicinalis is describedwhich directly acts one collagen-platelet interaction thus inhibitingplatelet activation and early platelet-platelet interaction.

Sofare, there has not been a positive example in the literature whichindicates that by using a screening approach that would excludeaggregation inhibitors as well as lytic proteins from a source ofnaturally occuring compounds one could identify new anti-adhesivemechanisms or compounds. However this strategy was used in thisinvention. Since at least six different platelet surface glycoproteinsare known to be involved in collagen adhesion and in addition severalplatelet derived compounds such as van Willebrand factor, fibronectinand thrombospondin have been shown to be involved as indirect mediatorsof collagen-platelet adhesion there has been little optimism in thebeginning toi identify a new adhesion inhibitors.

Nevertheless, this approach was used to screen Hirudo medicinalis salivaknowing that not all the documented or unkown vWF related inhibitors aswell as compounds directely acting on platelet receptors could beexcluded. Thus the result of the screening was a surprise: A new proteinnamed Saratin with anti-adhesive activity for plateles which can beisolated from tissues and secretions of well investigated leech of thespecies Hirudo medicinalis.

The present invention comprises the active polypeptide Saratin isolatedfrom the leech Hirudo medicinalis. The protein was isolated from salivaby a combination of pressurized dialysis and at least onechromatographic step like anionic exchange chromatography and at leastone reversed phase high performance chromatography (RP-HPLC) step. Thepressure dialysis step turned out to be absolutely critical during therecovery of Saratin from saliva, since the strong concentration ofsaliva helped to overcome the otherwise tremendous loss of bioactiveSaratin. The isolated Saratin binds strongly to several collagens andblocks the adhesion of platelets to collagen coated such surfaces in adose dependent fashion.

In order to optimize the screening cascade currently availabletechniques have been developed to distinguish platelet adhesion andplatelet aggregation: the ability of platelets to retard or stop flowthrough fibers, the contribution of platelets to in vitro clotformation, glass bead adhesion laboratories, or whole blood flowsthrough the filter and platelet adhesion of anticoagulated platelet-richplasma to filters composed of glass fibers or collagen under a regulatedpressure gradient.

The protein (named Saratin) is characterized by the amino acid sequencesdepicted in sequence (SEQ. ID. NO. 2) and is constituted from 103 aminoacids which make up a theorectical relative molecular weight ofapproximately 12068 dalton∓1 kDa The protein exhibits a unique primarystructure with no significant similarity to other previously describedsequences. The protein is rich in aspartic and glutamic acid whichcontribute to the low isoelectric point of of pH 3.7∓0.5 of the moleculeas measured by IEF-PAGE.

SDS-PAGE analysis demonstrated a strong shift in mobility upon reductionof the protein proir to electrophoresis, indicating posttranslationalmodifications. Sequencing of the polypeptide had revealed six cysteinemolecules which could make-up post-translational modifications of theprotein. Electrospray mass spectrometry of Saratin revealed an actualmolecular weight of 12061 indicating that up to three disulphide bondsare involved in the formation of the secondary structure of the nativeform of the protein.

The adhesion inhibitor according to the present invention is new becauseit differs from known aggregation inhibitors isolated from leechesespecially from Calin or LAPP in the molecular weight, isoelectric pointand amino acid sequence and biological activities.

The present invention provides as well isolated DNA comprising apolynucleotide encoding the leech derived platelet adhesion inhibitorhaving the amino acid sequence as shown for the protein. The nucleotidesequence representing the cDNA clone is shown in SEQ. ID. NO. 1,Position 1-63 of the nucleotide sequence represents a putative 21 aminoacid leader sequence and position 64-372 contains an open reading framecoding for a polypeptide of 103 amino acid residues and an amino acidsequence as shown for the mature protein in SEQ. ID. NO. 2.

The present invention also relates to recombinant vectors which includethe synthetic gene coding for the leech-derived platelet adhesioninhibitor of the present invention, and a host cell containing therecombinant vectors. Methods for recovering and isolating the expressedproteins have been based on tag-technologies or have been adapted fromthe purification scheme developed for the naturally occuring Saratin.Depending on the individual protocols used for extracellular orintracellular expression in yeast cells, insect cells, baby hamsterkidney cells and E. coli cells transformed with the appropriate vectorsthe steps for recovering the recombinant protein from the supernatant orsediments have to be adapted by techniques known to the expert.Excellent expression was found in E. coli as a host, where periplasmaticexpression was contributed by insertion of a peIB leader sequence.Products recovery from Escherichia coli (E. coli) was achieved (arround5 mg/l) after osmolysis and centrifugation; Saccharomyces cerevisiae (S.cerevisiae) (>10 mg/l culture broth) with the alternative yeast adoptedvector was used in a paralelled experiment. The secreted material wasisolated by centrigfugation. Purification was achieved by cross-flowfiltration and ion exchange chromatography. In other expressionapproches using either COS cells or CHO cells product expression wasarround 750 ng/ml. The purified recombinant material proved to be pureand homogeneous by electrophoretic and chromatographic analysis andidentical to saliva derived Saratin as demonstrated by amino acidsequencing and molecular mass determination.

The invention also comprises methods for purifying the active proteinfrom crude saliva of the leech and measuring its activity againstplatelets by static and dynamic assay methods as well as the use of thismethod to isolate rekombinant protein.

Techniques for the production of Saratin, include the Examples 6, 7, 8and 13 however the expression methods to be used are not restricted tothese examples. For instance transgenic mice, or other organisms,including other mammals, may as well be used to express Saratin.

Proteins of the present invention include variants which conserve theactivity of the disclosed sequences, including fragments or subunits,naturally occuring variants, allelic variants, randomly generatedartificial mutants and intentional sequence variations such as addingwhich conserve activity. Fragments or subunits refers to to any portionof the sequence which contain fewer amino acids than the completeprotein e.g. partial sequences excluding portions of the N- and/orC-termini of the complete protein.

The invention further covers hybrid proteins, such as fusion proteins orproteins resulting from the expression of multiple genes within theexpression vector, and may include a polypeptide having the specificactivity of a disclosed protein linked by peptide bonds to a secondpolypeptide. Notably other variants of the proteins of the presentinvention are included, especially any variants that differ from theisolated protein only by conservative amino acid substitution. Suchconservative amino acid substitutions are defined in Taylor et al., J.Mol. Biol., 1986, 188, 233.

Also included are methods for using the proteins to prevent or delay ofplatelet activation by inhibition of collagen-platelet interactions. Theprotein is useful in the prevention, prophylaxis, therapy and treatmentof thrombotic diseases. Unlike all these previously described proteins,which act at various surface proteins on the platelet, the protein fromthis invention has a unique mechanism of action. It binds tightly to thecollagen surface and one mechanism of action is given by the coverage ofspecific collagen sides no longer available for interactions and bindingof platelets. This type of new mechanism has the great advantage, thatthe platelets stay functionally intact during the application of theprotein, so that very low or even no bleeding tendency is expected fromthis treatment.

Another important area of use is the treatment of various surfaces withthe protein to render them non-adhesive for platelets and thereby createblood-compatible devices.

As indicated above, the polypeptides according to the present inventionare suitable as pharmaceutically effective compounds in parmaceuticalcompositions and combinations.

The pharmaceutical formulations according to the invention optionallymay comprise additional active incredients like Aspirin, anti-coagulantssuch as hirudin or heparin or thrombolytic agents such as plasminogenactivator or streptokinase.

The novel polypeptide according to the invention may formpharmaceutically acceptable salts with any non-toxic, organic orinorganic acid. Inorganic acids are, for example, hydrochloric,hydrobromic, sulphuric or phosphoric acid and acid metal salts such assodium monohydrogen orthophosphate and potassium hydrogen sulfate.Examples for organic acids are the mono, di and tri carboxylic acidssuch as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric,fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic,benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic and sulfonicacids such as methane sulfonic acid. Salts of the carboxy terminal aminoacid moiety include the non-toxic carboxylic acid salts formed with anysuitable inorganic or organic bases. These salts include, for example,alkali metals such as sodium and potassium, alkaline earth metals suchas calcium and magnesium, light metals of Group IIIA includingaluminium, and organic primary, secondary and tertiary amines such astrialkylamines, including triethylamine, procaine, dibenzylamine,1-ethenamine, N,N′-dibenzylethylene-diamine, dihydroabietylamine andN-alkylpiperidine.

As used herein, the term “pharmaceutically acceptable carrier” means aninert, non toxic solid or liquid filler, diluent or encapsulatingmaterial, not reacting adversely with the active compound or with thepatient. Suitable, preferrably liquid carriers are well known in the artsuch as steril water, saline, aqueous dextrose, sugar solutions,ethanol, glycols and oils, including those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil andmineral oil.

The formulations according to the invention may be administered as unitdoses containing conventional non-toxic pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles which are typical forparenteral administration.

The term “parenteral” includes herein subcutaneous, intravenous,intra-articular and intratracheal injection and infusion techniques.Also other administrations such as oral administration and topicalapplication are suitable. Parenteral compositions and combinations aremost preferably adminstered intravenously either in a bolus form or as aconstant fusion according to known procedures.

Tablets and capsules for oral administration contain conventionalexcipients such as binding agents, fillers, diluents, tableting agents,lubricants, disintegrants, and wetting agents. The tablets may be coatedaccording to methods well known in the art.

Oral liquid preparations may be in the form of aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives like suspending agents, emulsifying agents,non-aqueous vehicles and preservatives.

Topical applications may be in the form of aqueous or oily suspensions,solutions, emulsions, jellies or preferably emulsion ointments.

Unit doses according to the invention may contain daily required amountsof the protein according to the invention, or sub-multiples thereof tomake up the desired dose. The optimum therapeutically acceptable dosageand dose rate for a given patient (mammals, including humans) depends ona variety of factors, such as the activity of the specific activematerial employed, the age, body weight, general health, sex, diet, timeand route of administration, rate of clearance, the object of thetreatment, i.e., therapy or prophylaxis and the nature of the thromboticdisease to be treated, antiplatelet or anticoagulant activity.

Therefore, in compositions and combinations useful as antithrombotic ina treated patient (in vivo) a pharmaceutical effective daily dose of thepeptides of this invention is between about 0.01 and 100 mg/kg bodyweight, preferably between 0.1 and 10 mg/kg body weight According to theapplication form one single dose may contain between 0.5 and 10 mg ofthe thrombin inhibitor To achieve an anticoagulant effect inextracorporeal blood a pharmaceutically effective amount of theinventive peptides is between 0.2 and 150 mg/l, preferably between 1 mgand 20 mg/l extracorporeal blood.

It is also object of this invention to provide an implantable orextracorporal medical device for use in contact with body fluids inorder to render the device surface substantially thromboresistant,coated with an immobilized polypeptide as defined above and in theclaims. The polypeptide according to the invention is immobilzed on amedical device so as to render the surface biocompatible andthromboresistant. Such devices sometimes have surfaces properties whichtypically induce platelet aggregation, which is a disadvantage in theirintended uses in inplantable and extracorporeal devices in contact withblood or other body fluids. Examples for such devices which are commonlymade from plastics materials and synthetic fibres are protheses,artificial organs, ocular lenses, sutures, artificial vascular segments,catheters, dialysers, tubes and vessels carrying blood.

Posterior capsule opacification (PCO) is a common complication aftercateract extraxtion, despite the modern surgical techinques and lenseswhich are used for this procedure. PCO is caused by the proliferationand migration of lens epithelial cells across the posterior capsule thusreducing the visual acuity. Physical treatments as well as chemicallymodified lenses have been proposed to reduce formation of PCO. Heparinlens coating or topical heparin eyedrops have been used to reduce PCO,indicating that thrombogenic mechanisms are involved in the formation ofPCO.

Saratin has been shown to have significant advantages over heparin inpreventing and blocking thrombogenicity. It is therefore another featureof this invention to provide a coating comprising Saratin which reducesthrombogenicity of the lens material used for refractive anterior orposterior chamber ocular implants which may be surgically implanted intothe eye. This new type of coating avoids problems contributed bystimulated cell growth. In combination with other medicaments which arefor instance confering cell death, Saratin coating helps to completelyovercome posterior capsule opacification.

BRIEF DESCRIPTION OF THE FIGURES

Details of the figures are explained in examples 1 to 10.

FIG. 1

Separation of saliva components. Elution of Saratin is marked by *

a) shows the separation profile of saliva after DEAE anion exchange.Saratin fractions have been collected from peak 3 (Example 2).

b) shows re-chromatography of pooled fractions on Mono Q HR5/5. Sampleshave been collected from the last part of the major peak as indicates bybar (Example 2).

c) shows last chromatography step on semi-preparative analytical RP-HPLCof Saratin positive fractions collected from Mono Q HR5/5 (example 2).Active Saratin was recovered from the major peak (peak 3).

FIG. 2

SDS-PAGE of fractions collected from RP-HPLC. Saratin positive fractionsare marked * (Examples 2 and 3).

FIG. 3

E. coli expression vector for Saratin (Example 7).

FIG. 4

Baculo donor plasmid for Saratin (Example 8)

FIG. 5

Whole blood has been exposed to an artifical collagen surface andplatelets adhesion has been visualized by staining. Saratin has beenused as an inhibitor (Protein #607) Example 9

FIG. 6

Inhibition of platelet adhesion on collagen type III coated coverslipsunder shere conditions. Comparison of saliva and saratin. Example 9

FIG. 7

Saratin exhibits dose dependent inhibition of platelet binding adhesionto collagen type III coated coverslips under shere conditions. Example 9

FIG. 8

Yeast expression vector for Saratin (Example 13)

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Screening Adhesion Inhibitors

Platelet adhesion to collagen has been the functional background fourscreening saliva components. In addition four additional tests availablefor the assesment of various actions of antithrombotic drugs such as theAZOCOLL assay, amidase activity assay, vWillebrand dependent bindingassay and platelet aggregation assay have been used to excludefunctional properties which idealy would not be linked to an adhesioninhibitor. While most of this assays used here are standard assays, theplatelet adhesion assay had to be modified to fitt our specific needs.In short: Horm collagen (Nycomed) has been coated to 96 well plates(Nunc) by using acidified collagen at a concentration of 20 μg/ml andincubating the plates overnight. After washing the plates 3 times withPBS the residual surface of the wells have been blocked with 1% BSA.Platelets isolated freshly from human citronylated blood have been addedsimultaneously with fractions derived from the individual column steps.When required pre-activation of platelets has been performed byincubating platelets prior to use in TBS in the precence of bivalentmagnesia ions. Raw saliva, standardized to a total protein concentrationof 200 μg/ml, has been used as a standard to compare for inhibitoryactivity. The platelet inhibition assay turned out to be highlysucceptible to buffer changes and elevated salt concentrations. Sinceall the samples have been processed by ion-exchanger chromatography thedirect testing of the fractions in the inhibition assay turned out to becomplicated and unreliable. Therefore all samples to be tested have beenapplied to a Centricon based concentration step which simultaneouslyreduced ionic strenght and supports concentration prior to measurement.

EXAMPLE 2

Purification of a Natural Inhibitor

The invention used saliva collected from H. medicinalis which is knownto contain a number of bioactive proteins such as hirudin, elastaseinhibitors, collagenases and platelet aggregation inhibitors such ascalin (Munro, R. et al.; Blood Coagulation and Fibrinolysis, 1991, 2,179-184) and LAPP (Schaffer, L. W. et al.; Arterioscler. Thromb., 1993,13, 1593-1601). Besides this characterized proteins the majority of theroughly eighty proteins detectable by SDS-PAGE are still unkown. In thepresent invention the fractionated saliva and the screening strategy asdescribed in Example 1 was used in order to screen for the novelproteins that interferes directely with platelet collagen interaction.

Separation of an adhesion inhibitor activity from raw saliva turned outto be a critical task, mainly due to the irreversible loss of most ofthe adhesion inhibitory activity in the first chromatographic step.Additives such as 12% ethanol and divalent cations as recommended byMunro, R. et al did not improve the situation. However the high saltconcentration of the saliva in combination with the low overall proteinconcentration (190-250 μg/ml) asked for an initial concentration orbuffer exchange step. Thus several strategies for enrichment,concentration or buffer exchange were explored. Traditional dialysis ledto the complete loss of activity. Most of the other standard techniquessuch as ion exchangers, affinity columns, size exclusion (loss wasirrespective of the column resin) failed independent of the nature ofthe separation technology or buffer and addditives used. Surprisingly itturned out, that pressurized dialysis of 500 ml saliva was a successfulmethod to concentrate the saliva proteins (about 30-40 times) andsimultaneously get ride of the unwanted buffer components. Unexpectedlythe saliva raw material processed in this way was an ideal startingmaterial for further purification and the recovery of bioacitivityanti-adhesive components out of the saliva was no longer a real problem.Since cation exchangers or affinity colums turned out ot be aninsufficient step in purification, weak anion exchanger such asDEAE-Fastflow or EMD-DEAE-Fractogel were used. 12% ethanol and divalentcations have been tested, however the results were similar with orwithout this additives. Further optimization of the chromatographicsteps led to a sequence of DEAE-column, Mono Q-column and as a finalstep an reversed phase RP18 column. Optimization of the chromatographicconditions have been performed on a BiaCore chromatographic system usinganalytical columns available from Pharmacia. The gradients used tooperate the DEAE-column, Mono Q-column and RP18 colums are given inFIGS. 1 a, b, c. The BiaCore based separation has been scaled up byusing FPLC techniques. The optimized running conditions have beendirectly converted into the a semi-preparative scale of separation byfollowing the instruction of the manufacturer with the exception of theRP18 column step which was maintained with Biacore technique in order tominimize loss of purified material. Recovery of purified protein fromthe last chromatographic RP-step was done by speed vaccuumcentrifugation. Samples collected from the last RP-step have beencollected and analysed by SDS-PAGE (FIG. 2) Subsequently samples havebeen resuspended in PBS and used to perform analytical as well asfunctional tests. Typically a yield of about 750 μg/l Saratin wasrecovered from unprocessed saliva.

EXAMPLE 3

Biochemical Characterization

Purification of Saratin as disclosed in Example 1 led to an essentiallypure protein with an apparent moleculare weight around 21 kDa shownunder reducing conditions in SDS-PAGE (FIG. 2) The complete amino acidsequence was obtained by direct sequencing of the first 48 amino acidsof the purified protein and was completed by the sequencing of severalinternal peptides generated via enzymatic degradation. The completeprotein sequence is disclosed in SEQ. ID. NO. 2

The protein is composed of 103 amino acids with an calculated molecularweight of 12067.9 and an actual molecular weight of 12061.9 as deducedby ESI-Mass spectrometry. This differences in theroretical and measuredmolecular weight indicates that all the six cysteins identified inSaratin are involved in the formation of S-S bridges. This finding issupported by the observation of a strong change in mobility of theprotein when chromatographed in SDS-PAGE under reducing or none reducingconditions. Futhermore the protein is rich in acidic acids such as Gluand Asp. Isoelectric focussing using IEF-PAGE (Immobiline) techniquerevealed an isolelectric point of pH 3.7∓0.5. In a comparative study wehave used the purified leech protein as a reference and compared it withthe physicochemical properties of recombinat Saratin derived frombaculo, yeast and E. coli expression. All three protein turned out to beidentical in their properties. Characterization of protein by SDS-PAGEvisualized by Coomassie (FIG. 2) or silver-staining and/or Western blotanalysis revealed, that the protein was homogeneous and in a noneglycosylated form.

EXAMPLE 4

mRNA Preparation and cDNA Synthesis

RNA was prepared from the medicinal leech, Hirudo medicinalis; usingguanidinium thiocynate method. mRNA was purified from total RNA using“Oligotex mRNA kit” (QIAGEN).

cDNA was synthesized using “Marathon cDNA Amplification kit” (CLONTECH).The DNA sequence encoding Saratin was initially amplified using PCRoligonucletide primers according to the instruction of the supplier.After cDNA synthesis, a universal adaptor was ligated to both ends ofthe cDNA. The sequences of the universal adaptor and the universalprimers AP1 or AP2 was chosen according to the instructions of thesupplier of said kit.

EXAMPLE 5

Amplification and Isolation of the Saratin Gene by PCR

Degenerate primers have been synthesised based on the immediateN-terminus of the reverse translated Saratin amino acid sequence.

These primer01 and primer02 have been designed to hybridize specificallyto the 5′-end of the Saratin cDNA. The primer design was based on thereverse translated amino acid sequence of the experimentaly determinedeight N-terminal amino acids of the purified Saratin protein. The twoprimers primer01 and primer02 were synthsized to keep the degeneracy aslow as possible and to give the primers at the critical 3′-end a stretchof 8 basepairs of perfect match with the Saratin cDNA sequence in orderto ensure efficient and specific amlification of the template cDNA.According to the DNA degenerate alphabet (IUPAC code) R=A or G; M=A orC; Y=C or T; and N=A or G or C or T.

A 3′-RACE PCR reaction was carried out using a mixture of the primer01and primer02 and one universal primer AP1 or AP2. PCR products werecloned in TA cloning vector pCR2.1 or pCR Script SK(+) vector andsequenced. After sequencing several of the 3′-RACE PCR fragmentsobtained, the Saratin gene sequence was obtained, with the exeption ofthe 5′-untranslated region, the signal peptide sequence and the sequencecoding for the first 8 amino acids from the N-terminus of the matureprotein. In order to obtain this missing Saratin cDNA sequenceinformation, a 5′-RACE experiment was performed using a gene specificprimer from the middle of the Saratin cDNA and one of the universalprimers AP1 or AP2. After sequencing several of the resulting 5′-RACEPCR fragments, the complete Saratin gene sequence was obtained.Amplifying the Saratin gene by PCR using gene specific, non-degenerateprimers from both the 3′-end and the 5′-end of the Saratin gene, a fulllength of Saratin gene was obtained.

DNA Sequencing was performed with more then 15 different PCR clones.However only one significant change which caused amino acid sequencechange in only one clone was found. It is very likely that this changewas caused by PCR. Another five silent changes which do not cause aminoacid sequence change were found. Thus it is unlikely that these changeswere caused by PCR, because the same changes were found in differentclones.

The Saratin gene ORF is 372 bp and contains a 21 amino acid signalsequence and a 103 amino acid sequence coding for the mature protein.The amino acid sequence deduced from the PCR clone was found to beidentical to the sequence obtained by sequencing of the natural salivaderived protein.

EXAMPLE 6

Expression in COS Cells and the Detection of Expressed Protein

In order to express Saratin gene in mammalian cells such as COS or CHO,Saratin gene was cut from the vector pCR Script SK(+) using XhoI+XbaIand cloned into mammalian cell expression vector pCI-neo (Promega).pCI-neo was chosen because it contains T7 and T3 promoter for in vitroexpression and neomycin resistant gene for G418 selection, and can beused in both COS and CHO cells.

In addition to the signal sequence and mature protein sequence, theinsert contains a Koz. sequence at the 5′-end for efficient translationand the his-tag MRGS(H)₆ at the 3′-end (C-Terminal) for detection ofprotein expression, purification and concentration. The plasmidconstructure was named pNC-31.

pNC-31 plasmid DNA was used for the transfection of COS cells. The COScells growing at log phase were washed twice with PBS and dissolved inPBS at a concentration of 1×10⁷/ml. Then 12 μg plasmid DNA (less than 50μl in H₂O or TE buffer) was added in 0.7-0.8 ml COS cells suspension andmixed in a electroporation cuvette. Electroporation was performed at 1.9kv, 25 μFD for 10 min and transfered to a 90 mm plate. After adding 8 mlDMEM medium containing 10% FCS and antibiotics the cells were grown forthree days. The supernatantes and the cells have been used for furtherisolation of protein and detection. The expressed protein was detectedby western blotting method using anti-MRGS(H)6 antibody. Purificationwas performed using chelators such as NTA or imido acetic acidimmobilized on a column matrix and modified with metal ions such as Co,Ni, or Cu.

EXAMPLE 7

Construction of the E. coli Expression Vector and Expression

Because of the variable codon usage in different biological systems,some codons are used very rarely in E. coli. In order to enableexpression an optimized version in E. coli the gene had to be convertedto E. coli codon usage according to standard procedures.

The expression in E. coli was performed using a modified version of theplasmid pASK75, which carries the tet promoter region. (Skerra, A., etal. Gene, 1994, 151, 131-135). In short: The modification was made bycloning a new linker between the XbaI and the Hind III sites (FIG. 3).The new linker contains the ompA leader sequence, another multiplecloning site and a 6×His-tag instead of the strep-tag.

To construct the expression vector for Saratin it was necessary tointroduce 5′ Cla I and 3′ Eco47III restrictionsites by PCR method.

Therefore the 5′-primer03 and the 3′ primer04 have used. The PCR productfirst was cloned into the PCR II vector system (Invitrogen) andsequenced.

In a second step the Saratin gene was cloned into the modified pASK75vector using the rectrictionsites 5′ClaI and 3′ HindIII.

After expressing and proving the activity of this recombinant Saratin ina second PCR reaction the His-tag was removed and the start codon of theSaratin gene was directly fused to the omp A leader sequence. The5′-primer05 and the 3′-primer06 for this PCR reaction are given in thesequence listing.

As an example for the expression in E. coli the expression vector pRG72(FIG. 3), which contains the structural gene of Saratin fused to theompA leader sequence, was transformed into W3110 competent cells. Cellshave been induced after they have been grown to mid-log phase. 1 hourthereafter the recombinant Sarastatin could be clearly detected.

EXAMPLE 8

Construction of the Baculo Donor Plasmid and Expression

For expression of Saratin in the Baculo virus expression system theBac-To-Bac™ Baculovirus Expression System from Gibco Life Technologieswas used. To get a selection system the Honeybee melitin leader sequencewas fused to the Saratin gene and to introduce the restriction sites5′BamHI and 3′KpnI one single PCR reaction was carried out using the5′-primer07 and the 3′-primer08. The corrresponding PCR product wascloned into the PCR II Vector (Invitrogen) and sequenced. Then theMelitin-Saratin fusion was cloned into the pFastBac vector using therestriction sites 5′BamHI and 3′KpnI resulting in pTD13 (FIG. 4).Generation of recombinant baculoviruses and Saratin expression wasperformed with the Bac-To-Bac Expression System. The donor plasmid pTD13was transformed into DH10Bac competent cells which contain the bacmidwith a mini-attTn7 target site and the helper plasmid. The mini-Tn7element on the donor plasmid ca transpose to the a mini-attTn7 targetsite on the bacmid in the presence of transposition proteins provided bythe helper plasmid. Colonies containing recombinant bacmids wereidentified by disruption of the lacZ gene. High molecular weightmini-prep DNA is prepared from selected E. coli clones containing therecombinant bacmid, and this DNA was then used to transfect insectcells. Detailed descriptions are included in the instruction manual ofthe expression kit.

EXAMPLE 9

Platelet Adhesion to Collagen Under Flow Conditions (Dynamic Assay)

In the platelet adhesion assay, whole human blood is perfused through aparallel flow chamber to examine the adhesive activity of platelets to acollagen coated coverslips under high shear flow (simulating in vivoarterial conditions) as originally described by Sakariassen et al (Meth.Enz., 1988, 169, 37-70). Human placental collagen, type III (Sigma),solubilized in 50 mmol/L acetic acid was sprayed onto cleaned glasscoverslips (18 mm×18 mm) with a retouching airbrush. Coverslips werestored in PBS at 4° C. overnight.

Fresh whole human blood (anticoagulated with low molecular weightheparin; 20 U/ml) was prewarmed at 37° C. for 10 min before being used.Preparations of proteins according to this invention were pipetted ontocoverslips (30 μl per coverslip) and incubated for 10 min in a humidchamber at room temperature before being inserted into the perfusionchamber. The blood was allowed to perfuse through the chamber (at 37° C.for 5 min at a shear rate of 1300 s⁻¹.)

Subsequently, the coverslips are removed, washed in PBS and fixed in0.25% gluteraldehyde for 30 min., and thereafter stained withMay-Grünwald Giemsa. FIG. 9 shows a typical expamle. Extensive coveragewith stained platelets is seen in the untreated control surface. Acomparable surface pretreated with Saratin shows dramatically deminished(by 80%) platelet binding. Platelet adhesion was quantified with a lightmicroscope as shown in FIG. 5 (magnification×1000) coupled to acomputerized image analyzer (Leica). Results were expressed as thepercentage of the surface covered with platelets and plateletaggregates.

Comparison of saliva inhibitory activity has been compared to purifiedprotein as demonstrated in FIG. 6. Platelet adhesion on collagen typeIII coated coverslips at a shear rate of 1300 s−1 with crude saliva(#616) produced an inhibition of about 48% compared to control. Purifiedprotein (#607; Saratin) demonstrated a reduction in platelet depositionof about 81% at standardized protein concentrations. The inhibitioninduced by Saratin increases in a dose-dependent manner with higherconcentrations of purified protein as demonstrated in FIG. 7.

EXAMPLE 10

Immunization and Antibodies

With the first lot of highly purified natural protein available,immunization of animals has been started right away. Immune sera wereraised in rabbits and high titered reagents were available for furtherscreening. Additional antisera became available when the peptidesequence of the complete protein was available and three syntheticpeptides have been synthesized (amino-acid sequence 83-103, 13-30,58-69) coupled to KLH with a standard linker procedure and used forimmunization. Three sera directed to an N-terminal peptide segment andtwo sera specific for C-terminal peptides have established. With thehigh titered immune sera available it has been possible to establish anduse Elisa technology to monitor and quantitate purification of naturallypurified as well as recombinant protein. Thus the time consuming andrather work intensive platelet-inhibition assay could be replaced andwas applyed only to confirm the inhibitory potential of finally purifiedprotein.

EXAMPLE 11

Immuno-Assays for Estimation of Saratin Binding

Acidified Horm-collagen (Nycomed) has been used for the coating of96-well micro-titer plates (Nunc) 50 μL of the collagen solution (20μg/ml) has been used to coat plates over night. Prior to testing theplates have been washed three times with PBS and have been incubatedwith a BSA solution (1%) in order to prevent non-specific adhesion. 50μl Saratin has been added in serial delution and have been incubated forone hour. Plates were washed three times prior to the application ofanti-Saratin antibody for detection. After an additional one ourincubation step surplus antibody has been removed and a secondbiotin-labeled Antibody has been used for detection. Read-out has beenperformed via streptavidin-POD catalyzed colour reaction with substratessuch as ODB-tablets (Dako) measured at 490 nm.

EXAMPLE 12

Competition Assay for Screening Inhibitors

Recombinant tagged Saratin (His-tag) prepared as described in Example 7has been compared with native untagged Saratin for collagen binding.Plates coated with acidified Horm-collagen (Nycomed) have been preparedas decribend in Example 11. Detection was performed with rabbitanti-Saratin antibodies. The taggged and untagged Saratins showedidentical binding properties. Alternatively the untagged Saratin versionhas been modified by biotinylation (Pierce, biotinylation kit) andcompared to unmodified Saratin. Binding properties to collagen have beenidentical. Futhermore experiments have been performed using thebiotinylated Saratin to cross-compete with unmodified Saratin, peptides,saliva derived Saratin, complete saliva or antibodies directed toSaratin. Binding of the various “competitors” to collagen has beentested by estimating the binding of biotinylated Saratin using astreptavidine-POD conjugate and ODB-substrate reaction for detection.This assay comprising biotinylated Saratin was typically used for theestimation of Saratin concentration in saliva (750 μg/l), epitopemapping of Saratin directed antibodies, evaluation of bioactive Saratin,mutated Saratin. In order to explore the potential of the assay blockingas well as none blocking anti-Saratin antibodies raised with specificSaratin peptides have been used.

EXAMPLE 13

Yeast Expression Vector and Expression

The pichia multi copy expression system (Invitrogen) has been used as atypical example for yeast expression. The construction of the yeastexpression vector is shown in FIG. 8. For generation of the expressionvector for Saratin the PCR amplification method has been used togenerate restriction ends (5′ EcoR I, 3′Not I) compatible with theligation into the appropriate vector (pPIC9K). The following 5′-primer09and the 3′-primer10.

Before transforming the Pichia spheroplasts the expression vector hasbeen linearized with Sal I. Colonies have been screened for His⁺Mut⁺-positive mutants to ensure integration of the Saratin gene. Typicalgrowing conditions have been: 28-30° C., up to an optical density of OD2-6. Induction of expression was performed after resupending centrifugedcells in medium and addition of methanol up to a final concentration of0.5% and maintaining this condition for an extented time periode whichtypically would be 24 hours. After a typical fermentation periode of 6days the product was recovered from the supernatant and analysed bySDS-PAGE and ELISA.

1. A isolated polynucleotide encoding a Saratin polypeptide from H.medicinalis having a molecular weight of about 12,000±1 kD with thebiological activity of an inhibitor of collagen-dependent plateletadhesion, wherein said polypeptide binds to collagen thereby preventingthe adhesion of platelets to collagen.
 2. An isolated polynucleotide ofclaim 1, coding for a polypeptide of SEQ. ID. NO. 2, or a completecomplement thereto.
 3. The polynucleotide of claim 1, wherein saidpolynucleotide comprises a DNA sequence that is at least 80% identicalto that of SEQ ID NO: 1 over its entire length.
 4. An expression vectorcomprising a DNA sequence of claim
 1. 5. An isolated host cellcomprising the expression vector of claim
 4. 6. An expression systemcomprising a host cell of claim
 5. 7. A process for producing apolypeptide comprising culturing a host cell of claim 5 under conditionssufficient for the production of said polypeptide and recovering thepolypeptide from the culture supernatant or cell residue.